Unspliced XBP1 Counteracts β-Catenin to Inhibit Vascular Calcification

Abstract

Background: Vascular calcification is a prevalent complication in chronic kidney disease and contributes to increased cardiovascular morbidity and mortality. XBP1 (X-box binding protein 1), existing as the unspliced (XBP1u) and spliced (XBP1s) forms, is a key component of the endoplasmic reticulum stress involved in vascular diseases. However, whether XBP1u participates in the development of vascular calcification remains unclear. Methods: We aim to investigate the role of XBP1u in vascular calcification.XBP1u protein levels were reduced in high phosphate (Pi)-induced calcified vascular smooth muscle cells (VSMCs), calcified aortas from mice with adenine diet-induced chronic renal failure (CRF) and calcified radial arteries from CRF patients. Results: Inhibition of XBP1u rather than XBP1s upregulated in the expression of the osteogenic markers runt-related transcription factor 2 (Runx2) and msh homeobox2 (Msx2), and exacerbated high Pi-induced VSMC calcification, as verified by calcium deposition and Alizarin red S staining. In contrast, XBP1u overexpression in high Pi-induced VSMCs significantly inhibited osteogenic differentiation and calcification. Consistently, SMC-specific XBP1 deficiency in mice markedly aggravated the adenine diet- and 5/6 nephrectomy-induced vascular calcification compared with that in the control littermates. Further interactome analysis revealed that XBP1u bound directly to β-catenin, a key regulator of vascular calcification, via aa 205-230 in its C-terminal degradation domain. XBP1u interacted with β-catenin to promote its ubiquitin-proteasomal degradation and thus inhibited β-catenin/T-cell factor (TCF)-mediated Runx2 and Msx2 transcription. Knockdown of β-catenin abolished the effect of XBP1u deficiency on VSMC calcification, suggesting a β-catenin-mediated mechanism. Moreover, the degradation of β-catenin promoted by XBP1u was independent of glycogen synthase kinase 3β (GSK-3β)-involved destruction complex. Conclusions: Our study identified XBP1u as a novel endogenous inhibitor of vascular calcification by counteracting β-catenin and promoting its ubiquitin-proteasomal degradation, which represents a new regulatory pathway of β-catenin and a promising target for vascular calcification treatment.

A New Link in the Chain: Unspliced XBP1 in Wnt Signaling and Vascular Calcification

“…Building upon this background and their previous work, the current report from Yang et al 4 defines a novel role of XBP1u as a negative regulator of vascular calcification and osteogenic gene expression due to hyperphosphatemia using in vitro surrogates for vascular calcification, and as a physiological regulator of vascular calcification in vivo in the 5/6 nephrectomy and adenine-induced models of chronic kidney disease combined with hyperphosphatemia. The most interesting novel finding of this study is the identification of a Wnt and β-catenin regulatory function of XBP1u, whereby a physical interaction between the XBP1u C-terminal domain and β-catenin targets the latter for ubiquitin-proteasomal degradation.…”

Resistance Training Modulates Reticulum Endoplasmic Stress, Independent of Oxidative and Inflammatory Responses, in Elderly People

“…Translated XBP1s transactivates gene expression related to protein folding, ER quality control, and ER-associated protein degradation (ERAD) components [ 98 ], and regulates the transcription of genes involved in lipid and glucose metabolism [ 99 ] and immune responses [ 100 ]. However, although XBP1s is located in the nucleus, unspliced XBP1 (XBP1u) is found in the cytoplasm to not only involve in ER stress, but also help maintain other physiological functions [ 101 ], including: (1) the regulation of autophagy through FoxO1 interaction [ 102 ]; (2) cellular redox homeostasis via Nrf2-promoted transcription of heme oxygenase 1 (HO-1) in response to serine/threonine-protein kinase mTOR-mediated RAC-alpha serine/threonine-protein kinase (Akt1) phosphorylation [ 103 ]; (3) the promotion of tumorigenesis, leading to p53 ubiquitination/proteasomal degradation [ 104 ]; and (4) the regulation of vascular smooth muscle cells’ phenotypic transition, maintenance of vascular homeostasis [ 105 ], and the inhibition of vascular calcification [ 106 ].…”

Vascular calcification: Molecular mechanisms and therapeutic interventions

“… 71 Unspliced XBP1 could affect the expression of the osteogenic markers Runx2 and msh homeobox 2 (Msx2) by bounding directly to β‐catenin to influence the progression of VC. 72 ERS can be enhanced due to the upregulation of the protein levels of ATF4 and osteoblast markers (osteopontin and osteocalcin), 73 together with the downregulation of contractile markers (smoothelin, calponin, and SM22α), which resulted in the conversion of the VSMCs from contractile phenotype to osteogenic phenotype. Vice versa, when the levels of the contractile markers increased, the osteoblast‐like markers decreased, and VC is retarded with ATF4 knockdown.…”